STUDIES ON THE SUBSTRATE-SPECIFICITY OF NEUTRAL ALPHA-MANNOSIDASE PURIFIED FROM JAPANESE-QUAIL OVIDUCT BY USING SUGAR CHAINS FROM GLYCOPROTEINS

The substrate specificity of neutral alpha-mannosidase purified from Japanese quail oviduct [Oku, H., Hase, S., & Ikenaka, T. (1991) J. Biochem. 110, 29-34] was analyzed by using 21 oligomannose-type sugar chains. The enzyme activated with Co2+ hydrolyzed the Man-alpha-1-3 and Man-alpha-1-6 bonds from the non-reducing termini of Man-alpha-1-6(Man-alpha-1-3)Man-alpha-1-6(Man-alpha-1-3)Man-beta-1-4GlcNAc-beta-1-4GlcNac (M5A), but hardly hydrolyzed the Man-alpha-1-2 bonds of Man9GlcNAc2. The hydrolysis rate decreased as the reducing end of substrates became more bulky: the hydrolysis rate for the pyridylamino (PA) derivative of M5A as to that of M5A was 0.8; the values for M5A-Asn and Taka-amylase A having a M5A sugar chain being 0.5 and 0.04, respectively. The end product was Man-beta-1-4GlcNAc2. For the substrates with the GlcNAc structure at their reducing ends (Man5GlcNAc, Man6GlcNAc and Man9GlcNAc), the hydrolysis rate was remarkably increased: Man5GlcNAc was hydrolyzed 16 times faster than M5A, and Man9GlcNAc 40 times faster than Man9GlcNAc2. The enzyme did not hydrolyze Man-alpha-1-2 residue(s) linked to Man-alpha-1-3Man-beta-1-4GlcNAc. The [GRAPHICS] These results suggest that oligomannose-type sugar chains with the GlcNAc structure at their reducing ends seem to be native substrates for neutral alpha-mannosidase and the enzyme seems to hydrolyze endo-beta-N-acetylglucosaminidase, digests of oligomannose-type sugar chains in the cytosol.